Enolase is a component of the glycolysis pathway and a “moonlighting” protein, with important roles in diverse cellular processes that are not related to its function in glycolysis. However, small molecule tools to probe enolase function have been restricted to crystallography or enzymology. In this study, we report the discovery of the small molecule “ENOblock”, which is the first, nonsubstrate analogue that directly binds to enolase and inhibits its activity. ENOblock was isolated by small molecule screening in a cancer cell assay to detect cytotoxic agents that function in hypoxic conditions, which has previously been shown to induce drug resistance. Further analysis revealed that ENOblock can inhibit cancer cell metastasis in vivo. Moreover, an unexpected role for enolase in glucose homeostasis was revealed by in vivo analysis. Thus, ENOblock is the first reported enolase inhibitor that is suitable for biological assays. This new chemical tool may also be suitable for further study as a cancer and diabetes drug candidate.
Glycolysis is an ancient and highly conserved metabolic pathway that converts 1 mol of glucose into 2 mol of pyruvate. Free energy is released and used to form the highenergy-containing compounds adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis comprises 10 biochemical reactions, and each step is catalyzed and regulated by a different enzyme. Over the past 20 years, there has been increasing appreciation of the multiple roles glycolytic enzymes play in diverse cellular processes (reviewed in ref 1).
Cancer cells show increased dependence on glycolysis to produce ATP; a phenomenon known as the Warburg effect.2 This metabolic alteration is a fundamental difference between cancer cells and normal cells, offering a therapeutic strategy to selectively kill cancer cells using glycolysis inhibitors (reviewed in ref 3). It has also been shown that glycolysis inhibitors induce cancer cell death more effectively in a hypoxic environment, which occurs within developing tumors.4 Moreover, this hypoxic environment renders cancer cells less sensitive to other cancer drugs, such as cytarabine and doxorubicin.4 
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosures of cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls and details of the present invention are explained more clearly.